Studies from our VA-supported research and that of others demonstrate a critical role for the proinflammatory cytokine, tumor necrosis factor (TNF), in the development and progression of alcoholic liver disease (ALD). Alcoholic liver disease is associated with increased oxidative stress and impaired hepatocyte 26S proteasome function. Our preliminary data demonstrate that inhibition of 26S proteasome function can sensitize hepatocytes to TNF cytotoxicity, and higher doses of proteasome inhibitors directly induce hepatocyte death. Additionally, this apoptotic death involves the induction of lysosomal membrane permeability (LMP) and is mediated by lysosomal proteases (cathepsins B and D). Based on the increasingly evident role of cathepsins as key downstream mediators of apoptosis and our preliminary results, we postulate that chronic alcohol abuse with subsequent proteasome dysfunction leads to LMP and release of lysosomal cathepsins which induce hepatocyte cell death and further sensitize hepatocytes to TNF-mediated hepatotoxicity. Neutrophil infiltration is a hallmark of alcoholic hepatitis, and infiltrating neutrophils are thought to play an etiologic role in the liver injury in ALD. We recently demonstrated that inhibition of proteasome function in hepatocytes induces and markedly enhances the TNF-inducible expression of the pro-inflammatory and neutrophil chemotactic cytokine, Interleukin-8 (IL-8). It is our working hypothesis that chronic alcohol abuse mediated proteasome dysfunction is associated with increased hepatocyte IL-8 production, which significantly contributes to neutrophil infiltration and hepatic injury in ALD. The overall objective of this proposal is to elucidate the cellular and molecular mechanism(s) involved in proteasome dysfunction-mediated hepatic injury associated with ALD. The specific objectives of this proposal are to: 1) Determine the effects of proteasome inhibition and ethanol on sensitization to TNF hepatotoxicity, 2) Determine the role of lysosomal membrane permeability and cathepsins B and D in the proteasome dysfunction-mediated hepatocyte apoptosis and sensitization to TNF hepatotoxicity, and 3) Determine the effects of proteasome inhibition and ethanol on the hepatocyte expression of the critical neutrophil chemotactic CXC cytokine IL-8. Potential Impact on Veterans Health: Alcoholic liver disease is a leading cause of death from liver disease in the United States and in the Veterans Administration. In studies from the Veterans Administration, patients with cirrhosis and superimposed alcoholic hepatitis had > 60% mortality over a 4 year period of time, with most of those deaths occurring in the first month. Thus, the prognosis for this disease is more ominous than for many common types of cancer such as breast, prostate, and colon. The longterm goals of this laboratory are to define mechanism(s) for liver cell death in ALD that will provide new insights into this process and provide new areas for therapeutic intervention> This is especially important in ALD where there is no FDA-approved therapy. The potential clinical impact of this work is high. PUBLIC HEALTH RELEVANCE: The overall objective of this proposal is to elucidate the cellular and molecular mechanism(s) involved in alcohol and proteasome dysfunction-mediated hepatic injury associated with alcoholic liver disease (ALD). We expect that the results of our study will provide critical molecular insights into the pathogenesis of ALD, and provide a basis for clinically relevant paradigms for future development of potential therapeutic intervention(s) in patients with ALD.